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Linear-hairpin variable primer RT-qPCR for MicroRNA.

ABSTRACT: Here, we present a highly specific, sensitive and cost-effective system to quantify microRNA (miRNA) expression based on two-step RT-qPCR with EvaGreen detection chemistry, called linear-hairpin variable primer RT-qPCR. It takes advantage of the novel designed variable primer, which is initially designed to be linear, extending to form a hairpin structure and replacing the target miRNA for cyclic RT. Then the RT product is quantified by conventional EvaGreen based qPCR. The results show that this method has a dynamic range of 8 logs and the sensitivity is sufficient to directly detect down to 4 target miRNA molecules with a total analysis time of less than 2 hours. It is capable of discriminating between similar miRNAs, leading to an accurate representation of the mature miRNA content in a sample. The RT step can be multiplexed and the 8 miRNA profiles measured in 7 mouse tissues by this method show an excellent correlation with the commercial standard TaqMan RT-qPCR assays (r 2 = 0.9881).

SUBMITTER: Lan L

PROVIDER: S-EPMC6375362 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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Linear-hairpin variable primer RT-qPCR for MicroRNA.

Lan Lin L Guo Qiuping Q Nie Hemin H Zhou Chang C Cai Qingyun Q Huang Jin J Meng Xiangxian X

Chemical science 20181204 7

Here, we present a highly specific, sensitive and cost-effective system to quantify microRNA (miRNA) expression based on two-step RT-qPCR with EvaGreen detection chemistry, called linear-hairpin variable primer RT-qPCR. It takes advantage of the novel designed variable primer, which is initially designed to be linear, extending to form a hairpin structure and replacing the target miRNA for cyclic RT. Then the RT product is quantified by conventional EvaGreen based qPCR. The results show that thi ...[more]

PMID: 30842860

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